Project Grant R01NS134895

DYSFUNCTIONAL ORGANELLE-SPECIFIC AUTOPHAGY LEADS TO BRAIN ISCHEMIA-REPERFUSION INJURY - PROJECT SUMMARY: TRANSIENT CEREBRAL ISCHEMIA OCCURS IN VARIOUS CLINICAL SCENARIOS, INCLUDING TRANSIENT ISCHEMIC ATTACK (TIA), CARDIAC ARREST, HYPOVOLEMIC SHOCK, CARDIAC SURGERY, AND MEDICAL CONDITIONS RELATED TO BRAIN EDEMA OR BRAIN VASOSPASM. THE MAJORITY OF CEREBRAL ISCHEMIA SURVIVORS EXPERIENCE LONG-TERM NEUROLOGICAL SEQUELAE DUE TO BRAIN ISCHEMIA-REPERFUSION INJURY (IRI). THE OBJECTIVE OF THE PROPOSED RESEARCH IS TO INVESTIGATE A NOVEL MECHANISM OF THE DYSFUNCTIONAL MITOPHAGY AND THE SUBSEQUENT EXCESSIVE ACCUMULATION OF DAMAGED MITOCHONDRIA (MITO HEREAFTER) AFTER CEREBRAL ISCHEMIA. THESE DAMAGED MITO RELEASE APOPTOTIC FACTORS AND REACTIVE OXYGEN SCIENCES (ROS) CONTRIBUTING TO BRAIN IRI. MITOPHAGY, A SUBTYPE OF (MACRO)AUTOPHAGY, SELECTIVELY DELIVERS DAMAGED MITO TO LYSOSOMES FOR DEGRADATION. N-ETHYLMALEIMIDE SENSITIVE FACTOR (NSF) IS THE SOLE ATPASE FOR REGULATING CELLULAR MEMBRANE FUSION EVENTS. WE HAVE FOUND THAT NSF IS DEPOSITED INTO INACTIVE PROTEIN AGGREGATES IN NEURONS DESTINED TO DIE AFTER CEREBRAL ISCHEMIA. THESE NSF-DEFICIENT NEURONS PROGRESSIVELY ACCUMULATE WITH SUBSTANTIAL AMOUNTS OF DAMAGED MITO AND AUTOPHAGIC/MITOPHAGIC STRUCTURES, SUGGESTING THAT NSF IS A CRUCIAL LIMITING FACTOR FOR REGULATING MITOPHAGIC DEGRADATION ACTIVITY, I.E., MITOPHAGIC FLUX. FURTHERMORE, WE RECENTLY GENERATED A NOVEL NEURON-SPECIFIC NSF-DEFICIENT MOUSE LINE. IN THE ABSENCE OF BRAIN ISCHEMIA, NEURONS OF THE NSF-DEFICIENT MICE EXHIBIT A SUBSTANTIAL ACCUMULATION OF MITOPHAGIC STRUCTURES AND DAMAGED MITO, WHICH SUBSEQUENTLY LEADS TO AUTONOMOUS NEURONAL DEATH. THIS PHENOTYPE REPLICATES MAJOR NEUROPATHOLOGIC FEATURES OBSERVED IN WILDTYPE (WT) MICE AFTER CEREBRAL ISCHEMIA. MOREOVER, OUR RECENT STUDIES HAVE DEMONSTRATED THAT NSF-OVEREXPRESSION (OVEREXP) PROTECTED, WHILE NSF-DEFICIENCY EXACERBATED BRAIN IRI IN THE MOUSE MODEL. BASED ON THESE DISCOVERIES, WE PROPOSE TO TEST A NOVEL HYPOTHESIS STRONGLY SUPPORTED BY OUR DATA: NSF INACTIVATION RESULTS IN DYSFUNCTIONAL MITOPHAGY, LEADING TO AN EXCESSIVE BUILDUP OF DAMAGED MITO AFTER CEREBRAL ISCHEMIA. THESE DAMAGED MITO RELEASE APOPTOTIC FACTORS AND ROS, CONTRIBUTING TO BRAIN IRI. WE WILL TEST THIS HYPOTHESIS BY INVESTIGATING: (I) WHETHER, WHERE, AND WHY NSF INACTIVATION DISRUPTS THE MITOPHAGY PATHWAY AFTER CEREBRAL ISCHEMIA USING NSF- DEFICIENT, NSF-OVEREXP, AND WT MICE (AIM 1); AND (II) THE MECHANISM RESPONSIBLE FOR THE POST-ISCHEMIC NSF INACTIVATION AS WELL AS THE CORRESPONDING TREATMENT STRATEGIES USING PHARMACOLOGICAL AGENTS IN THE MOUSE CEREBRAL ISCHEMIA MODEL. THE PROPOSED STUDIES WILL HELP TO: (I) DETERMINE IF NSF INACTIVATION INDUCES BRAIN IRI VIA DISRUPTING MITOPHAGIC DEGRADATION ACTIVITY; (II) DISTINGUISH THE MITOPHAGY-RELATED AND -UNRELATED IMPAIRMENTS THAT ARE EXPLICITLY CAUSED BY NSF INACTIVATION FROM THOSE AFFECTED BY NSF-INDEPENDENT EVENTS; AND (III) DISCOVER THE MECHANISM AND TREATMENT STRATEGIES FOR ALLEVIATING NSF INACTIVATION AFTER CEREBRAL ISCHEMIA. THESE STUDIES WILL PROVIDE THE NECESSARY FOUNDATION FOR DEVELOPING THERAPEUTICS TO RESTORE THE MITOPHAGIC DEGRADATION ACTIVITY AFTER CEREBRAL ISCHEMIA.